The mechanisms responsible for cellular iron homeostasis are increasingly implicated in degenerative disease and aging. This is exemplified by Friedreich ataxia (FRDA), a neurodegenerative and cardiac disease caused by a deficiency of frataxin, a mitochondrial iron-binding protein. We were the first to report that frataxin binds iron. We have shown that frataxin self-assembles and functions as both a Fe(ll)- chaperone to diverse iron-binding proteins, and as a Fe(lll)-storage molecule with ferroxidation and mineralization activities that enable iron detoxification. We propose to test the following model: (i) The main function of frataxin is to detoxify redox-active iron in the mitochondrial matrix; (ii) frataxin accomplishes this by binding labile Fe(ll) imported from the cytoplasm or released in the matrix from superoxide-induced oxidation of [4Fe-4S] clusters; (iii) frataxin detoxifies labile Fe(ll) by promoting its insertion into iron-sulfur clusters and heme or its oxidation and incorporation into a stable mineral that is inactive in radical-generating reactions; (vi) while frataxin monomer only serves as a Fe(ll)-donor, assembly gives frataxin both the surface plasticity to interact with different iron-binding proteins and the structure to oxidize and store surplus iron. Using a collection of mutant frataxin proteins, we will dissect the mechanisms that enable frataxin to self- assemble (Aim 1), control iron toxicity (Aim 2), and donate Fe(ll) to different proteins (Aim 3). Frataxin variants with specific defects in self-assembly, iron uptake, and protein-protein interactions will be studied in S. cerevisiae cells with different endogenous sources of oxidative stress (Aim 4). Screens for genetic suppressors and synthetic lethal interactions of frataxin mutations will be used to identify proteins that act on frataxin improving its function or proteins that work with frataxin to detoxify mitochondrial iron (Aim 5). Our goal is to characterize the anti-oxidant function of frataxin and discover new determinants of oxidative stress resistance. RELEVANCE TO PUBLIC HEALTH The ability to handle iron efficiently and safely is critical to cell survival. By elucidating the underlying mechanisms, our work will help to identify fundamental sources of oxidative damage, degenerative disease, and unhealthy aging, and to develop preventive strategies and effective treatments.